Effects of arginine on intestinal epithelial cell integrity and nutrient uptake

Mi Xia†, Lulu Ye†, Qihang Hou and Qinghua Yu*Veterinary College, Nanjing Agricultural University, Weigang 1, Nanjing, Jiangsu 210095, People’s Republic of China

(Submitted 20 May 2016 – Final revision received 27 August 2016 – Accepted 10 October 2016 – First published online 14 November 2016)

AbstractArginine is a multifaceted amino acid that is critical to the normal physiology of the gastrointestinal tract. Oral arginine administration has beenshown to improve mucosal recovery following intestinal injury. The present study investigated the influence of extracellular arginine concentrationson epithelial cell barrier regulation and nutrition uptake by porcine small intestinal epithelial cell line (IPEC-J2). The results show that reducingarginine concentration from 0·7 to 0·2mM did not affect the transepithelial electrical resistance value, tight-junction proteins (claudin-1, occludin,E-cadherin), phosphorylated extracellular signal-regulated protein kinases (p-ERK) and mucin-1 expression. Furthermore, reducing arginineconcentration stimulated greater expression of cationic amino acid transporter (CAT1), excitatory amino acid transporter (EAAT3) and alanine/serine/cysteine transporter (ASCT1) mRNA by IPEC-J2 cells, which was verified by elevated efficiency of amino acid uptake. Glucose consumptionby IPEC-J2 cells treated with 0·2mM-arginine remained at the same physiological level to guarantee energy supply and to maintain the cell barrier.This experiment implied that reducing arginine concentration is feasible in IPEC-J2 cells guaranteed by nutrient uptake and cell barrier function.

Key words: Arginine: Intestinal epithelium: Mucosal barrier: Nutrient absorption

The intestinal mucosal epithelium acts as a physical barrier topathogens(1). The mucosal barrier is composed primarily of mucusand an epithelial cell membrane(2). Changes to the mucosal barriercan affect the absorption of both exogenous and endogenousnutrients(3). Therefore, the integrity of the mucosal barrier plays acrucial role in nutrition. Porcine small intestinal epithelial cell line(IPEC-J2) is an intestinal columnar epithelial cell line that wasisolated from the mid-jejunum of a neonatal piglet. The IPEC-J2cell line provides researchers with a unique tool: specificity forswine-based diseases and nutritional studies. Given the highhomology of porcine and human intestinal structure and functionand presumably similarity of enterocyte structure and function,studies using IPEC-J2 cells may provide valuable insights to theinteraction between nutrients and the human intestinal mucosa(4).Arginine is an essential amino acid in neonates, synthesised by

enterocytes and required for protein synthesis and maximumgrowth(5,6). It is the precursor of many important biological mole-cules such as proteins, nitric oxide (NO), ornithine and polyamine.Arginine signalling is important in the intestine, as it improves thestructure and function of the intestinal mucosa(7). Dietary argininealso promotes the growth of piglets, as well as the development ofintestine(8). More recently, it has been shown that supplementalarginine administration before a wound injury in IPEC-J2 cellscould increase cell migration in a dose-dependent biphasic mannervia NO and focal adhesion kinase-dependent mechanisms(9,10).In addition, a recent study demonstrated that arginine treatment

increased protein synthesis in IPEC-J2 cells via the rapamycin(mTOR)/ribosomal protein S6 kinase (p70S6K) pathway(11).

Most published studies focus on the effect of arginine on growthperformance. However, little is known about the effect of theconcentration of arginine on the intestinal epithelial cell barrier andnutrition uptake in vitro, given that more amino acid than neces-sary will cause waste and increase environment pressure, and high-quality protein is short in China. In the present study, we aimed toexplore the influence of reduction of arginine concentration (0 and0·2mM) in IPEC-J2 on cell integrity, as well as nutrient absorption.

Methods

Cell culture

In brief, IPEC-J2 cells were seeded on collagen-coated Millicellfilter inserts with a 0·4-μm pore size (Corning). IPEC-J2 cellswere cultured and maintained in Dulbecco’s modifiedEagle’s medium/nutrient mixture F-12 (DMEM-F12) (Life) sup-plemented with 10% fetal bovine serum (Gibco) andpenicillin–streptomycin (100mg/ml; Invitrogen). When theIPEC-J2 cells reached confluence (transepithelial electricalresistance (TEER)≥1000Ω× cm2)(12), cell culture medium waschanged to new medium supplemented with arginine at varyingconcentrations (arginine-free, 0·2 or 0·7mM-arginine).

Abbreviations: ASCT1, alanine/serine/cysteine transporter; CAT1, cationic amino acid transporter; EAAT3, excitatory amino acid transporter; GLUT2, glucosetransporter type 2; IPEC-J2, small intestinal epithelial cell line; p-ERK, phosphorylated extracellular signal-regulated protein kinases; SGLT1, sodium/glucosecotransporter 1; Slc, solute carrier; TEER, transepithelial electrical resistance; TJ, tight junction.

* Corresponding author: Q. Yu, fax +86 2584398669, email yuqinghua1981@163.com

† These authors contributed equally to this work.

British Journal of Nutrition (2016), 116, 1675–1681 doi:10.1017/S000711451600386X© The Authors 2016

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Transepithelial electrical resistance assay

TEER, a measure of tight-junction (TJ) integrity, is measured byan epithelial tissue volt-ohmmeter (Millicell ERS-2; MilliporeCorporation)(13). TEER was calculated from the measuredpotential difference between the apical and basolateral sides ofthe cell layer by an epithelial tissue volt-ohmmeter. All cellswere measured in real time (on the 3rd, 5th, 7th, 9th, 11th and12th day). Resistance is expressed as Ω× cm2.

Western blot analysis

The IPEC-J2 cells were collected and lysed in radio-immunoprecipitation assay (RIPA) lysis buffer (SunShine Bio).After centrifugation at 10 000 g and 4°C for 10min, the proteinconcentration in the supernatant was determined using a TecanInfinite 200 Pro (Tecan). All samples were adjusted to the sameprotein concentration, diluted in 4× loading buffer to a finalvolume of 2·5ml and heated in boiling water for 10min. Aftercooling on ice, the solution was used for Western blot analysis.Equal amounts of sample aliquots were subjected to 12%

SDS-PAGE and transferred to polyvinylidene difluoride (PVDF)membranes (Millipore) at 90V for 100min using the Bio-RadTransblot apparatus (Trans-blot Turbo system; Bio-Rad). Themembranes were blocked in 5% fat-free milk in TBST (Tris-buffered saline, 0.5% Tween 20) for 2 h and were then incubatedovernight with sodium/glucose cotransporter 1 (SGLT1) (SantaCruz; 1:800), glucose transporter type 2 (GLUT2) (Santa Cruz;1:1000), claudin-1 (Santa Cruz; 1:1000), occludin (Abcam;1:1000), E-cadherin (Abcam; 1:500), phosphorylated extracellularsignal-regulated protein kinases (p-ERK) (Bioworld Technology;1:1000), tubulin (Abcam; 1:1000) or β-actin (Vazyme Biotech;1:5000) antibodies at 4°C with gentle rocking.After washing five times with TBST, the membranes were

incubated at room temperature for 2 h with horseradish-peroxidase-linked secondary antibody (Vazyme Biotech;1:6000). Finally, the membranes were washed with TBST andthen developed using Supersignal West Dura Extended Dura-tion Substrate (Pierce) according to the manufacturer’s instruc-tions. The images were detected by chemiluminescence(Applygen). Western blots were quantified by measuring theintensity of the correctly sized bands using Quantity One-4.6.2(Bio-Rad). The ratio of the intensities of the protein bands ofinterest v. the housekeeping protein was calculated for eachfilter, and the ratios from different Western blot filters were usedto determine protein abundance.

Extraction of RNA and gene expression analysis in thejejunum using real-time PCR

Total RNA of IPEC-J2 cells was extracted from the cells usingTrizol reagent (Invitrogen). RNA integrity was assessed by 1%agarose gel electrophoresis and staining with 10mg/ml ethi-dium bromide. First-strand complementary DNA synthesis wasperformed with the quantitative reverse transcription (QRT)reverse transcriptase (Vazyme Biotech). Real-time PCR wasconducted using the 7500 Real-Time PCR System (ABI) andEvaGreen qPCR MasterMix-Low Rox (Abm). PCR amplification

was conducting using the following conditions: 95°C for 10min,followed by thirty-five cycles of 95°C for 30 s and 60C for 34 s.Each sample and negative controls (no template) were run intriplicate. The data were analysed using equation 2�ΔΔCT . ThemRNA expression of solute carrier family 1 member 4 (Slc1A4)(alanine/serine/cysteine transporter; ASCT1), solute carrierfamily 1 member 1 (Slc1A1) (excitatory amino acid transporter;EAAT3) and solute carrier family 7 member 1 (Slc7A1) (cationicamino acid transporter; CAT1) was analysed. β-Actin was usedas a housekeeping gene for data normalisation (Table 1).

Amino acid detection in cell culture medium using L-8900,a highly efficient amino acid analyzer

The amount of amino acids made by IPEC-J2 cells was measuredafter 12 or 24h of incubation in a glucose production medium(pH 7·4) supplemented with different arginine concentrations.For each sample, 200μl of medium was deproteinised with 200μlof trichloroacetate, mixed on vortex type mixer and centrifugedimmediately (4°C, 15min, 10000 rpm) to remove precipitatedprotein. The supernatant was purified with a 0·22-μm filter. Wemeasured amino acid concentrations with an automatic amino acidanalyzer (model L-8900; Hitachi) that separates amino acids bycation-exchange chromatography. Each amino acid was detectedspectrophotometrically after a post-column reaction with ninhydrinreagent. Reproducibility was verified by measurements of anamino acid mixture standard solution (Type AN-II; Wako)(14).

Glucose assay to measure glucose content of the medium

The amount of glucose in the culture medium was measuredafter IPEC-J2 cells were incubated in the glucose productionmedium supplemented with different arginine concentrationsfor 24 h. Glucose was detected using a Glucose Assay Kit(Rsbio), according to the manufacturer’s instructions.

Statistical analysis

Arithmetic means with their standard errors are presentedtogether. Statistical analyses were performed using SPSSStatistics 17.0 (IBM SPSS). The data were analysed usingone-way ANOVA and were given as the mean values.Significance was determined by * P< 0·05 and ** P< 0·01.

Table 1. Primers used for real-time PCR

Genes Primers Sequences (5'–3')* Size (bp) Tm (°C)

β-Actin Forward AGATCAAGATCATCGCGCCT 171 60Reverse ATGCAACTAACAGTCCGCCT

Slc1A1 Forward CCAGCAGTACAAAACCACGC 167 59Reverse AGCCCAGGACATTTATGCCG

Slc1A4 Forward TTCATTCTTCCCATCGGGGC 138 60Reverse CGTGGCTGTCACTAGGATGG

Slc7A1 Forward TCTGAACGTCCGTCAGTTCG 134 60Reverse GGAGGAGGGTCCTGTGATCT

Tm, melting temperature; Slc1A1, solute carrier family 1 member 1; Slc1A4, solutecarrier family 1 member 4; Slc7A1, solute carrier family 7 member 1; NCBI,National Center for Biotechnology Information.

* Sequences from the NCBI database.

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Results

Effects of arginine concentrations on small intestinalepithelial cell line integrity

Fig. 1(a) shows the cell culture schematic diagram. The TEERvalue of IPEC-J2 cells dropped significantly when arginine wasremoved from the cultural medium (Fig. 1(b)). However, the TEERin 0·2 and 0·7mM-arginine groups was maintained at high values.Moreover, the claudin-1,occludin and E-cadherin are importantcomponent proteins of TJ, which control the intestinal epitheliumparacellular pathway(15,16). Compared with 0·7mM-arginine, treat-ment with 0·2mM-arginine in the medium did not affect the proteinexpression levels of claudin-1, occludin and E-cadherin (Fig. 1(c)).However, the p-ERK expression level was also not affected,compared with 0·7mM-arginine groups (Fig. 1(d)). We also did notdetect a significant difference in mucin-1 expression by IPEC-J2cells in the 0·2 and 0·7mM-arginine groups (Fig. 1(e)). However,the expression levels of TJ proteins and mucin-1 were all sig-nificantly decreased in the arginine-free group (Fig. 1(c) and (e)).

Effects of arginine concentrations on CAT1, EAAT3 andASCT1 mRNA expression in small intestinal epithelialcell line

The lumenal plasma membrane of absorptive cells containsat least three amino acid transporters, one each for acidic,

basic and neutral amino acids(17). Compared with0·7mM-arginine in the medium, reducing the arginineconcentration in the cell culture medium enhanced themRNA expression levels of amino acid transporters. We foundthat the abundance of CAT1, EAAT3 and ASCT1 mRNAexpression after treatment with 0·2mM-arginine increasedsignificantly when compared with the other two groups(Fig. 2).

Effects of arginine concentrations on amino acid uptakeby small intestinal epithelial cell line

The uptake of the basic amino acids (lysine and histidine) byIPEC-J2 cells treated with 0·2mM-arginine was increased sig-nificantly at 12 and 24 h when compared with IPEC-J2 cellstreated with 0·7mM. However, the uptake of lysine, histidineand arginine IPEC-J2 cells in arginine-free medium wasdecreased significantly compared with the other two groups(Fig. 3(a) and (b)). We also found that the uptake of the neutralamino acid threonine was also increased under 0·2mM-argininein the medium (Fig. 3(c) and (d)). Our results revealed that theuptake of the acidic amino acid (aspartic acid) by IPEC-J2 cellswas significantly decreased in response to arginine at con-centrations ranging from 0·7 to 0mM at 12 and 24 h (Fig. 3(e)and (f)).

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Fig. 1. Effects of arginine levels on small intestinal epithelial cell line (IPEC-J2). (a) Experimental settings to study the barrier function of IPEC-J2 cells in a culturesystem. The schematic depicts the monoculture system, where IPEC-J2 cells were cultured with Dulbecco’s modified Eagle’s medium/nutrient mixture F-12(DMEM-F12). When the IPEC-J2 cells reached confluence, they were cultured in new medium supplemented with different arginine concentrations (arginine-free, 0·2 or0·7mM-arginine). (b) Transepithelial electrical resistance (TEER) of arginine-free, 0·2mM-arginine or 0·7mM-arginine-cultured IPEC-J2 cells from 3rd to 12th day aftermedia replacement. (c) The protein expression levels of occludin, E-cadherin and claudin-1 were detected by Western blot analysis. (d) The protein expression levels ofphosphorylated extracellular signal-regulated protein kinases (p-ERK) were also detected by Western blot analysis. (e) Mucin-1 (Muc1) mRNA expression was detectedby real-time PCR. Values are means (n 4), with their standard errors. Results are from four different experiments. * P<0·05 and ** P<0·01 as determined by ANOVA.

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Fig. 2. Effects of arginine levels on CAT1, EAAT3 and ASCT1 expression in small intestinal epithelial cell line (IPEC-J2). Cells were cultured for 24 h in arginine-freeDulbecco’s modified Eagle’s medium (DMEM) containing 0·2 or 0·7mM-arginine. CAT1 (a), EAAT3 (b) and ASCT1 (c) mRNA expression levels were detected byreal-time PCR. Values are means (n 4), with their standard errors. Results are from four different experiments. ** P< 0·01 as determined by ANOVA.

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Fig. 3. The effect of arginine levels on amino acid uptake by small intestinal epithelial cell line (IPEC-J2). Cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) without arginine, or containing 0·2 mM-arginine or 0·7 mM-arginine for 12 or 24 h, respectively. The level of absorption of basic amino acid ( , lysine; , histidineand , arginine) at 12h (a) and 24h (b); neutral amino acids ( , valine; , tyrosine; , phenylalanine; , threonine; , isoleucine and , leucine) at 12h (c) and 24h(d); acidic amino acids ( , aspartic acid) at 12h (e) and 24h (f) were detected by L-8900, a highly efficient amino acid analyzer. Values are the means (n 4), with theirstandard errors. Results are from four different experiments * P<0·05 and ** P<0·01 as determined by ANOVA.

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Effects of arginine on glucose transport in small intestinalepithelial cell line

Intestinal glucose absorption is mediated by the Na-dependentGLUT SGLT1, and the facilitated glucose transporter GLUT2provides a glucose exit into the bloodstream at the basolateralside(18). Our results showed that compared with the 0mM-arginine group the 0·2mM-arginine group had significantlyincreased SGLT1 and GLUT2 expression in the IPEC-J2 cells(Fig. 4(a) and (c)). However, there was no significant differencein Na+-dependent glucose absorption capacity among the threegroups (Fig. 4(b)). Cells treated with 0·2mM-arginine couldmaintain the glucose concentration in the basolateral side,compared with cells treated with 0·7mM-arginine. However, theglucose content at the basolateral surface of IPEC-J2 cellstreated with arginine-free medium was significantly decreasedcompared with the other two groups (Fig. 4(d)).

Discussion

Arginine stimulates protein synthesis and enhances cell survival(11).In the intestine, arginine could protect the mucosal barrier by

reducing intestinal permeability and improving intestinalstructure and function(19,20). In the present study, we found thatarginine is necessary for maintaining the mucosal barrier,which was demonstrated by the TEER value dropping rapidly inarginine-free medium. However, 0·2 and 0·7mM-argininemedium could maintain similar TEER values. This phenomenonis consistent to the previous findings in IPEC-1 cells, whichshowed that the number of cells and protein synthesisdid not differ between the 350 and 500 µM-arginine-treatedgroups(21).

TJ and adherens junction are important in the maintenance ofepithelial integrity. Claudin-1 and occludin protein are twoimportant components of TJ, whereas E-cadherin is the majortransmembrane protein of the adherens junction(22,23). Forma-tion of the adherens junction leads to assembly of the TJ(24). Thefunction of arginine in cell integrity was further verified byclaudin-1 and mucin-1 expression, both of which weredecreased markedly in arginine-free medium. Compared withthe 0·7mM-arginine medium, a similar trend of claudin-1 andmucin-1 expressions was detected in cells treated with the0·2mM-arginine medium. The activation of p-ERK will disrupt

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Fig. 4. Effects of arginine levels on glucose transport by small intestinal epithelial cell line (IPEC-J2). Cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) without arginine, or containing, 0·2mM-arginine or 0·7mM-arginine for 12 or 24 h, respectively. Sodium/glucose cotransporter 1 (SGLT1) (a) and glucosetransporter type 2 (GLUT2) (c) protein expression was detected by Western blot analysis. Glucose transport levels were calculated from the glucose concentrationdifference between the apical (b) and basolateral (d) sides of the IPEC-J2 cells using a Glucose Assay Kit. Values are the means (n 4), with their standard errors.Results are from four different experiments. * P< 0·05 as determined by ANOVA.

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the TJ structure(25,26). We found that the p-ERK expression levelin cells treated with 0·2mM-arginine in the medium was main-tained at normal level, compared with the 0·7mM group. Theseresults together demonstrated that arginine is necessary for themaintenance of IPEC-J2 cell integrity, and reducing arginineconcentration from 0·7 to 0·2mM did not affect cell integrity.Amino acids are indispensable for epithelium survival and its

biological functions. Amino acids are efficiently absorbed bysmall intestinal enterocytes through amino acid transporters(27).The transporters for amino acids in the small intestine can bedivided into basic, neutral and acidic amino acid transportersystems(28). Arginine could stimulate CAT-1-mediated arginineuptake and regulation of inducible nitric oxide synthase forgrowth, as observed in chick intestinal epithelial cells(29). In thisexperiment, we found that reducing arginine concentration to0·2mM could stimulate greater expression of Slc7A1 (CAT1),Slc1A1 (EAAT3) and Slc1A4 (ASCT1) mRNA when comparedwith the 0·7mM-arginine group or without arginine in themedium. The enhanced expression of amino acid transportersimplied efficient uptake of amino acids. This was furtherverified by the amino acid absorption in our experiments. Wefound that the 0·2mM-arginine group improved the absorptivecapacity of IPEC-J2 cells for essential and functional amino acids(lysine and histidine) at 12 or 24 h, which were mediated byCAT1. Although the absorption amount of arginine in 0·2mM

medium was not increased significantly and maintained at thesame level with 0·7mM medium, the arginine absorptionefficiency was actually increased, considering the low arginineconcentration in 0·2mM medium. Previous studies have shownthat threonine promotes mucin synthesis and augmentsintestinal structure(30). The increased threonine absorption in0·2mM-arginine medium in this study could explain the main-tenance of the TJ protein and mucin-1 mRNA expression levels.Previous findings demonstrated that dietary arginine supple-mentation could affect the absorption of other amino acids inthe small intestinal mucosa(20). In addition, branched-chainamino acids (valine, isoleucine and leucine) may be necessaryto maintain normal intestinal development and absorption ofamino acids(31,32). We found that the absorption of otherdetected amino acids (valine, tyrosine, phenylalanine,isoleucine, leucine and aspartic acid) in cells treated with0·2mM-arginine could also be maintained at normal levelscompared with 0·7mM-arginine in the medium, which isnecessary to protect the epithelial integrity. Under low argininelevels, high absorption efficiency may be a compensatorymechanism for amino acid uptake by IPEC-J2 cells to guaranteenormal physiological functions.Glucose is a major source of energy for body tissues, parti-

cularly for the small intestine in young animals(33). Increasedamino acid absorption, supported by the high amino acidtransporter expression, guarantees energy needs. We found thatglucose consumption at the free surface and glucose content atthe basolateral surface of IPEC-J2 cells treated with 0·2mM-arginine medium were similar to cells treated with the 0·7mM-arginine medium. Moreover, the protein expression of SGLT1and GLUT2 at 0·2mM-arginine did not change when comparedwith 0·7mM-arginine, which is inconsistent with glucose con-sumption. However, the expression levels of the SGLT1 and

GLUT2 proteins were decreased significantly in the arginine-free medium. One possible reason is that the appropriatearginine dose for promoting cellular function (e.g. barrierdevelopment and absorption of amino acids) requires moreenergy consumption. This needs to be further studied. Previousstudies have shown that amino acid and glucose transport couldbe modulated by NO(34). Interestingly, arginine is an essentialsubstrate for synthesis of NO. This may partly explain howdifferent arginine concentrations affect the transport of glucosein epithelial cells.

In conclusion, we demonstrated that arginine is necessary forIPEC-J2 cells to maintain the epithelial integrity. However,reducing the arginine concentration from 0·7 to 0·2mM did notaffect cellular integrity, as measured by TEER, or claudin-1 andmucin-1 expression. This maintenance of cell integrity was alsofurther verified by the stable p-ERK expression. Moreover,0·2mM-arginine stimulated amino acid absorption efficiency byincreasing amino acid transporter expression, which guaranteesa sufficient energy supply with stable SGLT1 and GLUT2protein expression. This experiment illustrated that reducingarginine concentration to 0·2mM is feasible in IPEC-J2 cells,which provides guidance in developing dietary formulas forpigs at low protein concentrations.

Acknowledgements

M. X. and L. Y.: study conception and design, performance ofthe experiments, data analysis and interpretation, manuscriptwriting; Q. H.: performance of the experiments, data analysis;Q. Y.: study conception and design, financial support, admini-strative support, data analysis and interpretation, manuscriptwriting, final approval of the manuscript.

This work was supported by the National Basic ResearchProgram of China (973 program 2013CB127302), NationalNatural Science Foundation of China (31502024), A ProjectFunded by the Priority Academic Program Development ofJiangsu Higher Education Institutions and State Key Laboratoryof Animal Nutrition Fund of China (2004DA125184F1410).

There were no conflicts of interest (financial, professional orpersonal) relevant to the manuscript.

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